"All rats were observed daily for clinical signs of toxicity. Body weight was recorded twice a week in all males and in the satellite group females, and twice a week during the premating period, on days 0, 4, 7, 11, 14, 17, and 20 of pregnancy and on days 0 and 4 of lactation in main group females. Food consumption was recorded twice a week in all males and in satellite group females and twice a week during the premating period, on days 1, 4, 7, 11, 14, 17, and 20 of pregnancy and on days 2 and 4 of lactation in main group females. Functional observation battery (FOB) in all animals was recorded once a week during the administration period, as follows: (i) home cage observation; posture, convulsion, and abnormal behavior, (ii) in-the-hand observation; ease of removal from cage and handling, fur and skin condition, eye ball, secretion from nose and/or eye, visible mucous membrane, lacrimation, salivation, piloerection, pupil diameter, and respiration, and (iii) open field observation; arousal, ambulation, posture, shivering, convulsion, rearing frequency, excreta, stereotypical behavior, and abnormal behavior.

Five animals in each group were subjected to the following observations and examinations unless noted otherwise. Sensory reactions for pupillary reflex, approximation reflex, tactile reflex, auditory reflex, pain reflex, righting reflex and width of the landing legs, grip strength of fore and hind limbs, and spontaneous motor activity were tested in main group males on day 37 of administration, in main group females on day 4 of lactation, and in satellite group males and females on day 37 of administration and on day 8 of the recovery period. Fresh urine was sampled from animals using a urine-collecting cage during the last weeks of the dosing and recovery periods. The 4-hr urine samples were collected soon after dosing under fasting (water was allowed ad libitum), and the 20-hr urine samples were collected, food and water being allowed ad libitum.

After 16-20 hr (overnight) of fasting, the main group of rats was euthanized by exsanguination under anesthesia on the day after the final administration in males and on day 4 of lactation in females, and satellite group rats were euthanized on the day of the completion of the recovery period. The external surfaces of the rats were examined. The abdomen and thoracic cavity were opened, and gross internal examination was performed. Blood samples were drawn from the abdominal aorta. Major organs were removed from all animals, and the brain, thyroid, thymus. heart, liver, spleen, kidney, adrenal glands, testis, epididymis were weighed. The numbers of corpora lutea and implantation sites were counted in all main group females. The testes and epididymides were fixed with Bouin's solution and in 10% phosphate-buffered formalin. Other organs were stored in 10% phosphate-buffered formalin. The cerebrum and cerebellum, pituitary gland, spinal cord, sciatic nerve, thyroid, parathyroid, adrenal glands, thymus, spleen, mandibular lymph nodes, mesenteric lymph node, heart, lung, trachea, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, bladder, testis, epididymis, uterus, seminal vesicle, sternum, and femur were histopathologically evaluated for five males and females in the control and the highest groups, and organs with macroscopically abnormal findings were also examined histopathologically. The organs for histopathological evaluations were processed routinely for embedding in paraffin, and sections were prepared for staining with hematoxylin-eosin. Test substance-related histopathological changes were found in the liver in males and females, and in the stomach in males; therefore, the liver in all animals and the stomach in all males were also examined histopathologically.

The 4-hr urine samples were tested for color, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen, and urinary sediment. Urinary sediment was stained and examined microscopically. The 20-hr urine samples were tested for osmotic pressure. Urine volume for 4-hr and 20-hr was measured. In the collected blood samples the red blood cell (RBC) count, hemoglobin, platelet count, and white blood cell count were measured. In addition, mean corpuscular volume (MCV), hematocrit, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte rate, and differential leukocyte rates were calculated. Prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen were determined. Blood chemistry was tested for alkaline phosphatase (ALP), total protein, albumin, albumin/globulin (A/G) ratio, total bilirubin, blood urea nitrogen (BUN), creatinine, glucose, total cholesterol, triglycerides, phospholipid, Na, K, Cl, Ca, inorganic phosphate, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactase dehydrogenase (LDH), and gamma-glutamyltransferase (y-GTP). In the main group, daily vaginal lavage samples of each female were evaluated for estrous cyclicity throughout the premating period. Each female rat was mated overnight with a single male rat of the same dosage group until copulation occurred or the 2-week mating period had elapsed. During the mating period, daily vaginal smears were examined for the presence of sperm. The presence of sperm in the vaginal smear and/or a vaginal plug was considered as evidence of successful mating. Once insemination was confirmed, the females were checked twice a day for signs of parturition from day 21 to day 24 of pregnancy. One female in the 0.1 mg/kg/day treatment group did not deliver and did not have implantation. Because of infertility, data for that female for the period corresponding to gestation were excluded from statistical analysis. Other females were allowed to deliver spontaneously and nurse their pups until postnatal day (PND) 4. The day on which parturition was completed by 17:00 was designated as PND 0. Litter size and numbers of live and dead pups were recorded, and live pups were sexed and individually weighed on PNDs 0 and 4. Pups were inspected for external malformations on PND 0. On PND 4, the pups were euthanized by exsanguination under anesthesia, and gross internal examinations were performed.

Statistical analysis of pups was carried out using the litter as the experimental unit. Mean and standard deviation in each dose group were calculated for body weight, food consumption, water consumption, number of feces, rearing frequency, width of the landing legs, grip strength, spontaneous motor activity, urine volume, hematological test results, blood biochemical test results, absolute and relative organ weights, estrous cycle length, length of gestation, numbers of corpora lutea and implantations, implantation index, total number of pups born, number of male and female pups, number of live and dead pups, live birth index, live pups and viability index on day 4 of lactation, and body weight of pups. These were analyzed with Bartlett's test or F-test for homogeneity of variance. If they were homogeneous, the data were analyzed using Dunnett's test or Student's t-test to compare the mean of the control group with that of each dosage group, and if they were not homogeneous, a Dunnett-type rank test or Aspin-Welch t-test was applied. The copulation index, fertility index, gestation index, sex ratio of pups, and data for sensory reactions of reflexes were analyzed with Yates' chi-square test. The 5% levels of probability were used as the criterion for significance."

F1 Male Crl:CD(SD) Rat